Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids

The principle of sequential injection (SI) was exploited to develop a fully automated pre-column derivatization procedure combined on-line to liquid chromatography (LC). Using SI-LC derivatization 14 amino acids were determined fluorimetrically in pharmaceuticals with o-phthaldialdehyde (OPA) as the derivatization reagent. The SI system was used for the handling of samples and reagents, on-line mixing and introduction to the LC injection system. Chemical (pH and reagents concentrations) and instrumental variables (sample and reagent volumes, reaction time and flow rate) were optimized to attain the highest reaction yield and detector signal. Reversed phase chromatographic resolution of 14 amino acids was achieved within 35 min using gradient elution. The automated operation of the coupled SI-LC system resulted in very satisfactory performance. The method was applied for the simultaneous determination of amino acids in pharmaceutical formulations.     

A mixed-bed, multi-derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection

Immobilized, polymeric reagents containing covalently attached tagging groups have been synthesized and reacted individually, off-line or on-line, pre-column in high performance liquid chromatographic (HPLC) detection. These reagents have also been combined into a single, mixed-bed reactor, useful for simultaneously preparing several derivatives from a single analyte, all at the same time. Each derivative possesses different chromatographic and detection properties, dependent on the nature of the original polymeric reagent containing the immobilized, tagging species. These particular reagents were designed to impart Ultraviolet/fluorescence, Ultraviolet/electrochemistry (oxidative/reductive or oxidative-hv-electrochemistry) to the final derivatives. Variations in the amounts/ratios of polymeric reagents contained in a single mixed-bed reactor will lead to varying ratios of the final derivatives. These can be predicted knowing the approximate reactivity of each polymeric reagent, percent derivatizations, and overall rates for each reagent towards a given substrate. In this first example of mixed-bed, polymeric reagents for improved derivatization approaches in chromatography, emphasis has been placed on simple amines or amine-like analytes. Multiple derivatives can be effectively used to improve the identification of an unknown analyte in a complex sample matrix, as well as to improve the detectability of that analyte. As one real world application, amphetamine in human urine was quantitated via on-line derivatizations with a mixed-bed reactor. With the least sampling work-up, the resulting sample solutions were directly injected into the on-line derivatization HPLC system for quantitation. The method was validated by single blind spiking experiments. The precision and accuracy were acceptable.     

Flow injection spectrophotometric determination of copper, iron, manganese, and zinc in animal feeds using a common manifold

A rapid and simple procedure was developed for the determination of copper, iron, manganese, and zinc in animal feeds using an identical flow injection spectrophotometric manifold but different chromogenic reagents and different detection wavelengths. Bis(cyclohexanone)oxalydihydrazone, formaldoxime, 1,10-phenanthroline, and xylenol orange were adopted as chromogenic reagents for Cu, Mn, Fe, and Zn, respectively. Detection conditions such as manifold parameters, buffer pH, reagent concentration, temperature, and acidity of sample solution were optimized. Analytical characteristics of the method and interference of metal ions commonly present in feeds were studied. By changing the reagents and detection wavelengths, which can be done quickly, the proposed low cost flow injection system can determine Cu, Fe, Mn, or Zn in the range of 0.5-10 mg/L with a sampling throughput of 120/h.     

A catalytic multi-pumping flow system for the chemiluminometric determination of metformin

A multi-pumping flow system for the chemiluminometric determination of the hypoglycaemic drug metformin was implemented. The developed methodology was based on the metformin-induced inhibition (metformin acts as a Cu(II) scavenger) of the catalytic effect of Cu(II) ions on the chemiluminescent reaction between luminol and hydrogen peroxide. The flow manifold configuration was based on the utilisation of multiple solenoid-actuated micro-pumps that were simultaneously accountable for sample/reagent introduction and reaction zone formation/propulsion, thus resulting in a fully automated, simple and highly selective multi-pumping flow system. A versatile sample manipulation allowed the establishment of distinct sampling strategies with low reagent consumption. The characteristic pulsed flow ensured an effective sample/reagent mixing leading to a better and faster reaction zone homogenisation and thus improved analytical signals. Linear calibration plots were obtained for metformin hydrochloride concentrations ranging from 5 to 15 mg L^–1 with a relative standard deviation lower than 2.0% (n=5). Detection limit was 0.94 mg L^–1, and the sampling rate was about 95 determinations per hour. The developed methodology was applied to the analysis of pharmaceutical formulations and the obtained results were in agreement with those furnished by the reference method with relative percentage deviations of lower than 1.5%.     

衍生化-气相色谱-三重四极杆质谱法测定泼尼松龙中联氨

泼尼松龙是一种广泛用于临床治疗的肾上腺糖皮质激素药物,其中联氨的残留会直接影响用药安全,但目前国内外还没有出台相应的法律法规和标准来管控药物中联氨的残留限值.联氨具有强极性和强还原性,理化性质很不稳定,易被氧化,又因缺少发色团,相对分子质量太小,检测起来难度很大,需引入一种衍生化试剂,降低其极性,生成相对分子质量较大且...     

Solid-phase analytical derivatization: enhancement of sensitivity and selectivity of analysis.

Analytical derivatizations enhance the sensitivity and selectivity of determinations for organic compounds. Classical techniques are often based on solution chemistry. Most modern sample preparation techniques, however, are based on solid-phase extractions. Solid-phase analytical derivatization bridges this gap and facilitates sample preparation by combining the isolation step with the derivatization. The solid-phase retains both reagents and derivatized analytes and often permits facile separation of excess reagent or selective elution of the desired products. The most recent solid-phase extraction techniques have been used in conjunction with analytical derivatization to automate the analysis. In this review, analytical derivatizations are presented as functional group analysis.     

A robust multi-syringe system for process flow analysis. Part 3. Time based injection applied to the spectrophotometric determination of nickel(II) and iron speciation.

A new software-controlled time-based system for sample or reagent introduction in process flow injection analysis was developed. By using a multi-syringe burette coupled with one multi-port selection valve, the time-based injection of precise known volumes was accomplished. Characteristics and performance of the injection system were studied by injecting an indicator in a buffered carrier. Two multi-syringe time-based injection (MS-TBI) systems were implemented: first, the injection of a sample in a multiple-channel manifold where the sample would sequentially merge and react with different reagents, and second, the sequential injection of several solutions (sample and reagents) into a particular flowing stream. The first system was applied to the spectrophotometric determination of nickel(II) in diluted samples from the acidic nickel ore leaching process, by using ammonium citrate as carrier, a saturated solution of iodine as oxidizing agent and alkaline dimethylglyoxime as chromogenic reagent. The sampling frequency attained was 57 h-1. Determinations on process samples compared well at the 95% confidence level with the reference values obtained by ICP-OES. The second time-based injection system was applied to the speciation of iron. Total iron and iron(II) concentrations were separately and sequentially determined using 1,10-phenanthroline in acetic buffer medium as reagent. The developed manifold allowed the optional use of two different carrier solutions, containing or not containing ascorbic acid, for performing the separate determinations. Also, in the sequential procedure, plugs of reducing carrier were alternatively intercalated before the sample injections used for total iron determinations. Sampling frequencies of 68 injections per hour were routinely used. Accuracy was assessed by analyzing synthetic known mixtures of Fe(III) and Fe(II) standard solutions. Recoveries of 98-100.5% with a maximum relative standard deviation of 3.6% were found. Results obtained for various samples of fertilizers agreed well with those attained by the standard batch procedure.     

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non‐derivatized amino acids and biogenic amines

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE‐enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2‐2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.     

Sample preconcentration with chemical derivatization in capillary electrophoresis. Capillary as preconcentrator, microreactor and chiral selector for high-throughput metabolite screening

New strategies for integrating sample pretreatment with chemical analyses under a single format is required for rapid, sensitive and enantioselective analyses of low abundance metabolites in complex biological samples. Capillary electrophoresis (CE) offers a unique environment for controlling analyte/reagent band dispersion and electromigration properties using discontinuous electrolyte systems. Recent work in our laboratory towards developing a high-throughput CE platform for low abundance metabolites via on-line sample preconcentration with chemical derivatization (SPCD) is primarily examined in this review, as there have been surprisingly only a few strategies reported in the literature to date. In-capillary sample preconcentration serves to enhance concentration sensitivity via electrokinetic focusing of long sample injection volumes for lower detection limits, whereas chemical derivatization by zone passing is used to expand detectability and selectivity, notably for enantiomeric resolution of metabolites lacking intrinsic chromophores using nanolitre volumes of reagent. Together, on-line SPCD-CE can provide over a 100-fold improvement in concentration sensitivity, shorter total analysis times, reduced sample handling and improved reliability for a variety of amino acid and amino sugar metabolites, which is also amenable to automated high-throughput screening. This review will highlight basic method development and optimization parameters relevant to SPCD-CE, including applications to bacterial metabolite flux and biomarker analyses. Insight into the mechanism of analyte focusing and labeling by SPCD-CE is also discussed, as well as future directions for continued research.     

Quantitative assessment of cysteine and cystine in peptides and proteins following organomercurial derivatization and analysis by matrix-assisted laser desorption ionization mass spectrometry

Protocols for the analysis of the sulfhydryl content in peptides and proteins using chemical derivatization by organomercurial reagents and analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have been developed. The number of reactive cysteine residues in peptides and proteins can be determined by exploiting the affinity and selectivity of organomercurial reagents for macromolecular thiols. Mass shifts observed in MALDI mass spectra obtained before and after cysteine derivatization with p-hydroxy-mercuribenzoate (pHMB) permit the number of free sulfhydryl groups to be determined. The pHMB derivative of each free cysteine residue provides a mass shift of 321 u, overcoming limitations in the mass resolution of MALDI time-of-flight mass spectrometry. Reactive cysteine residues in a macromolecule can be selectively derivatized by using a fivefold molar excess of pHMB reagent. Total sulfhydryl content (i.e., cysteine and cystine) can be determined after disulfide reduction. However, analyses for total cysteine content are more complex, requiring protein denaturation, cystine reduction, and sample purification before derivatization and analysis by MALDI-MS. Conditions for sample denaturation, alkyl-phosphine reduction, pHMB derivatization, and sample purification by analyte adsorption and desalting on protein transfer membranes, are described for cysteine/cystine analysis performed on microgram (10–200 pmol) quantities of somatostatin, insulin, hemoglobin, and β-lactoglobulin.     

Simple and rapid determination of hydrogen peroxide using phosphine-based fluorescent reagents with sodium tungstate dihydrate.

A simple batch method for the fluorometric determination of hydrogen peroxide using phosphine-based fluorescent reagents has been developed. A rapid, mild and selective derivatization reaction was achieved by adding sodium tungstate dihydrate to the reaction mixture of hydrogen peroxide and a phosphine-based fluorescent reagent. When 4-diphenylphosphino-7-methylthio-2,1,3-benzoxadiazole was used as a reagent, the derivatization reaction was completed after 2 min at room temperature. The calibration curve was linear between 12.5 and 500 ng hydrogen peroxide in a 10 microL sample solution. This method is accurate and has potential for on-line applications.     

Study of different parameters affecting the derivatization of acidic herbicides with trimethylsulfonium hydroxide to make them suitable for gas chromatography analysis

In this study, an orthogonal array design was applied to know the way different parameters affected the derivatization of some herbicides that are commonly applied in the soils. Herbicides formulated as esters have been reported to rapidly hydrolyse, in contact with soil, to their corresponding acids and phenols. What involves is that both forms need to be monitored. Acidic herbicides and phenols cannot be detected by gas chromatography (GC) due to their polarity and low volatility that cause peak asymmetry. Therefore, masking of these polar groups by eliminating the active hydrogen atom with derivatization to their corresponding esters/ethers is needed in order to yield products that possess enhanced volatility and improved GC properties. A lot of derivatization reagents have been proposed but trimethylsulfonium hydroxide (TMSH) was selected due to its easy and quantitative formation of methyl esters/ethers. It was observed that the addition of TMSH promoted not only esterification of acids/phenols but trans-esterification of the original non-hydrolyzed remaining esters to their corresponding methyl ones. As a result, methyl esters/ethers were the final product of both reactions. Different parameters were studied in the statistical design for both TMSH promoted reactions: type of solvent, pH, temperature and time of incubation. The amount of derivatization reagent was calculated to be high enough to ensure the complete derivatization of all compounds present in the sample. The reaction medium was shown as an important factor. The formation of some methyl esters/ethers decreased with increasing time and temperature because trans-esterification, being an equilibrium where the formation of smaller structures is promoted, was not enough shifted. However, the statistical analysis revealed that only the pH of the solution played an important role during the derivatization process. The presence of the anionic form of the acids appeared to be essential for derivatization, being diminished in strong acidic conditions. In addition, pre-heating was shown not to improve derivatization reaction, being easily carried on in the injector port of the GC system.     

Continuous flow derivatization system coupled to capillary electrophoresis for the determination of amino acids

A derivatization system coupled to capillary electrophoresis for the determination of amino acids using 1,2-naphthoquinone-4-sulfonate as a labeling agent is described. In this system, amino acids are derivatized on-line in a three-channel flow manifold for sample, reagent and buffer solutions. The reaction takes place in a PTFE coil heated at 80 degrees C. The resulting solution, which contains the amino acid derivatives, is introduced into the electrophoretic system by means of an appropriate interface. Subsequently, amino acid derivatives are separated at 25 kV using a 40 mM sodium tetraborate aqueous solution with 30% (v/v) isopropanol solution as a running buffer. The electropherograms are monitored spectrophotometrically at 230 nm. The method has been applied to the determination of amino acids in feed samples and pharmaceutical preparations. A good concordance of the predicted values with those given by a standard amino acid analyzer is shown.     

Indirect enantioseparation of alpha-amino acids by reversed-phase liquid chromatography using new chiral derivatizing reagents synthesized from s-triazine chloride

Ten chiral dichloro- and monochloro-s-triazines were prepared by the nucleophilic displacement of chlorine atom(s) in s-triazine chloride and its 6-methoxy derivative with different amino acid amides. Dichloro-s-triazines (DCTs) were used as CDRs for derivatization of alpha-amino acids under basic conditions at room temperature (30 degrees C) while derivatization with monochloro-s-triazine (MCT) reagents was carried out at 80 degrees C. The resultant diastereomers were separated on a reversed-phase C(18) column using mixtures of acetonitrile and aqueous-trifluoroacetic acid (TFA). The separation results for the two were compared. One DCT reagent was optimized for derivatization kinetics with respect to the effects of pH, reagent excess, temperature and reaction time on derivatization yield. In most of the cases, DCT reagents provided better separation of diastereomers in comparison to MCT reagents. One DCT reagent was also validated for limit of detection, linearity, recovery and robustness. Effects of structural modifications in reagents on chromatographic properties were investigated. Separation mechanism of diastereomers was proposed in light of both MCT and DCT reagents.     

Single reaction interface in flow analysis

The dual or multiple reaction interface concept, commonly associated to the distinct flow techniques, was replaced by a single interface concept, which do not no rely on the utilisation of a well-defined and compelling sample volume but only on mutual penetration of sample and reagent zones at a single reaction interface where both sample and reagent met together prior to detection. In the proposed approach basic principles of flow analysis, such as controlled dispersion and reaction zone formation, are not influenced by sample and reagent volumes but determined exclusively by the extension of the overlap of two adjoining quasi-infinite zones enhanced by multiple flow reversals and the pulsed nature of the flowing streams.The detector is positioned at the core of the flow manifold (not in the conventional terminal position), and repetitive flow reversals enable interface manipulations, including multi-detection of the entire reaction interface or the monitoring of the evolution of a pre-selected interface zone by using suitable reversal cycle times.The implementation of the developed approach was facilitated due to the configuration simplicity and operational versatility of multi-pumping flow systems. Its performance was evaluated by monitoring processes involving two or four-solution reaction interfaces.     

Improved coupled-column liquid chromatographic method for the determination of glyphosate and aminomethylphosphonic acid residues in environmental waters

An existing method for the determination of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in water has been improved. It is based on precolumn derivatization with the fluorescent reagent 9-fluorenylmethylcloroformate (FMOC) followed by large-volume injection in a coupled-column LC system using fluorescence detection (LC-LC-FD). The derivatization step was slightly modified by changing parameters such as volume and/or concentration of sample and reagents to decrease the limits of quantification (LOQ) of glyphosate and AMPA to 0.1 microg/l. Additionally, the use of Amberlite IRA-900 for preconcentration of glyphosate, prior to the derivatization step, was investigated; the LOQ of glyphosate was lowered to 0.02 microg/l. Drinking, surface and ground water spiked with glyphosate and AMPA at 0.1-10 microg/l concentrations were analysed by the improved LC-LC-FD method. Recoveries were 87-106% with relative standard deviations lower than 8%. Drinking and ground water spiked with glyphosate at 0.02 and 0.1 microg/l were analysed after preconcentration on the anion-exchange resin with satisfactory recoveries (94-105%) and precision (better than 8%).     

(2-Naphthoxy)acetyl chloride,a simple fluorescent reagent

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.     

Postcolumn derivatization liquid chromatography/mass spectrometry for detection of chemical-weapons-related compounds

Postcolumn derivatization for liquid chromatography/mass spectrometry (LC/MS) analysis was characterized for detection of some compounds related to chemical-weapons (CW) agents using an Atmospheric Pressure Chemical Ionization (APCI) source. The derivatizing reagents were added directly to the LC eluent flow, and the derivatization reactions occurred in the APCI source under typical operating conditions. The compound S-[2-(diisopropylamino)ethyl] methylphosphonothioic acid was methylated using the derivatizing reagent trimethylphenyl ammonium hydroxide (TMPAH). Methylphosphonic acid was doubly derivatized to form dimethyl methylphosphonate, although the signal for the derivatization product was very sensitive to the amount of TMPAH. Arsenic compounds related to the CW agent lewisite, including chlorovinyl arsonous acid and arsenic (III) oxide, were derivatized using 2-mercaptopyridine. The thiol group reacted readily with the arsenic (III) center and provided a significant improvement in sensitivity relative to the underivatized signal using APCI or electrospray ionization. Triethanolamine and ethyl diethanolamine were derivatized with benzoyl chloride, a commonly used LC derivatizing reagent for alcohols, to modify their mass spectra. Postcolumn derivatization using an APCI source gives an alternative for detecting some difficult-to-ionize compounds. It has the limitations that sensitivity was not always improved even though the major mass spectral peaks can be shifted; it is necessary to carefully select the reagent; and some reagents introduced strong interference peaks at specific masses in the spectrum and may suppress the ionization of some derivatized analyte ions. The reagent also produced contamination in the source, which had to be cleaned daily.     

Piezoelectric pumping in flow analysis: Application to the spectrophotometric determination of gabapentin

Solutions propelling devices are fundamental components of a flow-based analytical manifold. In this work different manifold configurations were implemented to evaluate the performance of multiple piezoelectric micro-pumps used as solutions insertion and propulsion devices. The micro-pumps are piezo-actuated micro-diaphragm pumps with passive check valves characterised by a small compact size and low power demands, and are able to produce reproducible flow rates of up to 4 mL min⁻¹. The flow rate is controlled by the frequency of the piezoelectric actuator (up to 20 Hz). As an additional feature, piezoelectric micro-pumps actuation generates a pulsed flowing stream that ensures a faster sample/reagent mixing contributing to improved reaction development.

The developed flow approach was assessed in the spectrophotometric determination of gabapentin in pharmaceutical preparations upon reaction with 1,2-naphthoquinone-4-sulfonate in alkaline medium. Distinct flow manifold configurations were designed for achievement of different solutions management. Linear calibrations plots for gabapentin concentrations of up to 150 mg L⁻¹, with relative standard deviations of less than 1.50% (n = 10) and a sample throughput of about 28 determinations per hour, were obtained.     

A multicommutation-based flow system for multi-element analysis in pharmaceutical preparations

A flow system exploiting multicommutation and multidetection is proposed for sequential determinations in pharmaceutical preparations. The feasibilities were demonstrated by the determination of zinc, iron, copper, calcium and magnesium without changing the flow set-up. The gravitational flow of the solutions was exploited for addition of different chromogenic reagents and sample aliquots, thus avoiding the use of a propulsion unit. Transient signals at different wavelengths were measured simultaneously employing a fiber-optic multichannel spectrophotometer. Coefficients of variation of 1.0, 1.5, 1.4, 2.5 and 2.0% were obtained for iron, zinc, copper, calcium and magnesium, respectively. The mean sampling rate for the five species was 60 determinations per hour. In comparison to continuous reagent addition systems, the consumption was up to 160-fold lower. Results for pharmaceutical preparations agreed with those obtained by Flame atomic absorption spectrophotometry (FAAS) at the 95% confidence level.